Skin is the largest organ of the body, covering the body's outer surface area. Skin is composed of two main layers: the surface epithelia or epidermis, containing epidermal cells as keratinocytes, and the underlying connective tissue layer or dermis, containing dermal cells as fibroblasts. Several and important functions are associated to the skin, for instance, the protection against injury and dehydration, acting as a barrier against infections, perceiving or detecting environmental stimuli, excreting diverse substances, regulating the body temperature, and helping to maintain the hydric balance. Substantially intact and healthy skin is needed, not only for the body welfare, but for its survival.
The skin health and integrity may be affected by congenital or acquired pathological conditions, either acute or chronic, for which the natural skin repair and regeneration processes may be insufficient. These conditions include burns, wounds, ulcers, infections, congenital diseases and/or abnormalities. Therefore, a non-appropriate condition and state of the skin is extremely harmful, since it affects the health condition and the organism existence. Those patients that have been affected by burns in a vast surface area of their bodies often require an immediate and extensive replacement of skin. In addition, some less life threatening conditions can be present, however considered as chronic conditions of skin, for instance as occurs for passive hyperemia, the diabetic ulcers or decubitus ulcer, which may result in more severe conditions, if no treatment is applied, particularly since those patients exhibiting these conditions suffer an underlying pathology. On the other hand, the morbidity and mortality drop-off in this kind of patients depends of the appropriate and effective restoration of the skin structure and function.
Skin substitutes may be used for treating these or other conditions. The desirable properties for skin substitutes are availability and handling, low rejection degree, high adherence degree, highly independent from a skin donor, relative easiness for being produced from a skin biopsy of a minimal size, and the feature of a cost-effective production and use. Several methods for elaborating skin substitutes have been assayed, which satisfy some or all these requirements, but with varied success degrees.
However, a predominant design for a skin substitute, which satisfactorily is able of regenerating all the skin structures and functions, is not available yet. Only, a full thickness skin graft is able to virtually restoring all the normal non damaged skin structures and functions. Large and extensive information is available about commercial devices or technical solutions for solving this skin graft problem, nevertheless these do not provide the qualities and advantages of the development described in the present invention.
US 2003/0170892, describes a device comprising a cell-free biocompatible reticulated matrix, wherein a first (layer) of dermal cells are cultured, and over these cells, epidermal cells are grown. Further, it describes the method for producing said device, useful for applications in skin wounds, in therapeutic treatment and/or for the in vitro assay of human skin. Providing similar structures and functions to a normal healthy skin, as a barrier function.
The above development exhibits clear differences and disadvantages regarding the present invention, since the described implant corresponds to a device comprising cells grown in the surface and in clearly differentiated layers. Furthermore, due to the design and the method for producing said device, the required time for applying the same is longer than 24 hours, considered from the moment when the dermal cells culture is available for being seeded over the matrix.
CN 1562392 describes a method for preparing an artificial skin in an active bi-layer supported on a collagen sponge, which is cultured in a bioreactor with a fibroblasts suspension for generating the dermis and on top of this, epidermal cells are seeded. Structural differences with the corresponding graft of the present invention, can be clearly distinguished since this document mentions a layer type of graft exhibiting cells on the surface, wherein the seeding method is less efficient, more complex, more expensive and takes longer time for reaching similar results. The same differences regarding the present invention are observed for the patent application CN 1468634, wherein an artificial skin implant with a double film is described, with superficial growth of epidermal cells over a fibroblasts-PGA structure.
U.S. Pat. No. 6,733,530 describes a device wherein autologous keratinocytes are grown over a biocompatible substrate, which is pre-seeded with allogenic or autologous dermal fibroblasts, which can be applied over an artificial skin substrate grafted to the receptor patient. The layered and superficial structure of the device, and the method for preparing the same, described in this document, makes the present invention a different device with clear advantages regarding the applications of the same.
In a same way to the preceding cases, in the patent CA 2497895 is described a fibrin cell support to form cell cultures. It is possible to establish cell cultures on this support, such as keratinocytes cultures. In a disclosed embodiment, it is possible to incorporate cells in the fibrin gel structure. The above description considerably differs from the present invention device, since the present invention corresponds to a gel-matrix-cells integrated device, which exhibits better handling characteristics as it comprises a biocompatible support available as the porous matrix, which contributes to device strength, resistance and flexibility. In fact, the presence of a biocompatible support does not form part of what is described in the document, since the fibrin gel is used in combination with a preliminary treatment of the wound with artificial skin (Integra™). Furthermore, in the document the product is applied as a paste or spray on the wound, which implies great difficulties for fixing the fibrin gel to the wound and for being properly accepted by the grafted patient. Accordingly, combined additional or supplementary treatments are needed, in order to allow the graft fixing to the wound, since the fibrin gel slides from the application site. In the present invention no fixing or additional treatments are required in order to maintain the device on the application site and to start the regeneration of the damaged skin. Although in the reference is mentioned the incorporation of polymers, these function only as fibrin carriers, furthermore these are not incorporated into the fibrin gel, as in the present invention. The above provides a different support with a closer in situ integration of the matrix-gel, corresponding to one of the present invention contributions.
In the same way the patent application WO02078721 describes a system for providing an autologous in situ implant, wherein a keratinocytes and fibroblasts suspension containing fibrin, are mixed with a thrombin, fibrinogen and collagenase solution. As in the latter case, the reference differs from the present invention, as it is not related to an integrated device comprising a biocompatible support as a porous matrix. At the same time, in the present invention the strength, handling and adherence properties are improved and optimized, regarding the technical solution disclosed in the document WO02078721. Furthermore, the physical qualities of strength and the in situ preparation, makes this product difficult to handle and store, since it corresponds to a cell suspension which is applied in the site to be treated (wound or damaged tissue), exhibiting adherence difficulties such as described for the previous document.
The main purpose of the skin substitutes is to save the patient's life, providing a coating or barrier, which prevents the skin dehydration and infection. A second objective is to allow for a functional healing, which additionally should be cosmetically acceptable.
Different cell types and material combinations, which have been evaluated for producing cutaneous substitutes, are nowadays commercially available. Most of said commercial products are formed by allogenic cells-containing matrix, generally from neonatal foreskin, which exhibits the advantage of containing a higher number of stem cells for keratinocytes, potential mitogenic properties, an exacerbated metabolism and minimal antigenicity(1,2).
One of the known most simple cutaneous substitutes is EPICEL®, which is composed by autologous keratinocytes arranged on a paraffined gauze, which is a non biodegradable material. Said cells are obtained from a full thickness biopsy from the injured subject, the cells are subsequently grown over a layer of murine fibroblasts, which have previously been irradiated. Thus, stratified keratinocytes layers are achieved, containing from two to eight layers(3). In this system, rejection is totally avoided and the cells are permanently incorporated into the tissue. However, it is fragile and with poor cosmetic ability of maintaining an acceptable appearance in the patients. Some available studies indicate that implanting stratified keratinocytes (differentiated) is not the most suitable approach, since the higher differentiating degree and the lower proliferating ability. It is preferable, such as is provided by the present invention, to implant cells with the least number of in vitro passages.
A retrospective study carried out in a 30 patients group, exhibiting burns that cover 78% of the body surface, treated with EPICEL®, an extremely high survival rate (90%) was informed(4). Nevertheless, the higher difficulty of this treatment is related to its cost and logistics.
Other known product, based in autologous cells is Laserskin®, which is indicated for the treatment of second-degree deep burns and for chronic ulcer. This product consists of a biodegradable estherified hyaluronic acid matrix, with laser made micro-perforations in order to allow the keratinocytes settlement and proliferation. Said autologous keratinocytes are obtained from a biopsy and are directly cultured in the mentioned matrix. Laserskin®, is only available in Europe. The efficiency in treating the diabetic foot ulcer has been demonstrated for this product; however no controlled clinical trials are available. This device can be used combined with Hyaff™ a support containing dermis fibroblasts, wherein the fibroblasts can be autologous or allogenic. These products correspond to devices wherein cells can be grown within their matrix, not only in the surface, nevertheless, these do not correspond to an in situ integrated system formed by matrix-gel cells as the one of the present invention.
On the other hand, Celaderm® is a commercially available product, this product contains metabolically active foreskin-derived heterologous keratinocytes which however are unable of proliferating. This product has been used for treating chronic ulcer; and as a further advantage, it can be cryo-preserved(6). Some available studies reveal the effectiveness of this product in burns, however not compared with autografting(7). This product exhibits significant differences regarding the present invention, as it does not involve an integrated system.
Other known product is Dermagraft®, an approved substitute for treating diabetic foot ulcer. In this case, fibroblasts are obtained from neonatal foreskin and cultured on a polyglactine matrix, during approximately three weeks. In this term, cells secrete matrix proteins, providing a tridimensional in situ matrix which serves as a dermis substitute. The product is delivered cryo-preserved and requires thawing and washing to be used(8).
Other product is Transcytee® (originally known as Dermagraft-TC®) containing non-viable fibroblasts on a silicone-covered nylon net. Said product is prescribed as a temporary cover or coating for burn wounds which have been split by surgery, as an alternative for cadaver skin(9,10).
Other allogenic product is the one known as LSE, primary used in the treatment of the diabetic foot and the venous leg ulcers(11). The LSE corresponds to a cutaneous substitute composed by a collagen matrix with viable fibroblasts and a stratified epidermal layer. One difference regarding the present invention is that it corresponds to an integrated device without forming stratified or differentiated epithelia.
The OrCel® product is very similar to the previous one with the difference that the dermal matrix is a porous, crosslinked type II collagen sponge, instead of a gel. The sponge is non symmetrical, in a way that a side is covered by a layer of an acid soluble atelocollagen gel, for sealing the macroscopic pores. Fibroblasts are grown on or within the porous side of the collagen sponge, while the keratinocytes are cultured in the non-porous side, covered by the gel of this collagen matrix. The cells seeded matrix is kept submerged for inhibiting differentiation and stratification of keratinocytes. The time and ratio for the fibroblasts and keratinocytes seeding are designed for controlling the cellular density and the cytokines expression in the final product. It has been seen that the co-culture of fibroblasts and keratinocytes exhibits synergic effects over the production levels of some cytokines and growth factors(12-13).
The PolyActive™ product, uses polyethylene oxide/polybutylphtalate (PEO/PBT) and may use the own patient fibroblasts for the dermis, and the own patient keratinocytes cultured for the epidermis. Finally, Integra™ which uses a collagen-glycosaminoglycan matrix (GAG) providing a non cellular or acellular dermal component, and which may use a thin autograft or other devices containing cells.
The tissue engineering discipline, intended to the generation of a solution for burned patients, or with disability scars or cutaneous ulcers, is an area of great scientific interest. Numerous scientific publications can be found, wherein products are described, which contain autologous or heterologous cells, in combination with some different nature matrix.
In general, the market known and existing products exhibit differences and disadvantages regarding the present invention. The main differences are that said devices do not correspond to an in situ integrated device with a fibrin gel and a biocompatible polymer. Among the disadvantages of the state of the art devices, the content of bovine nature components can be found; therefore, it should be considered that these products soon will no longer be used, due to the appearance of prion contamination cases in USA, the need of long incubating or preparation periods, and the differentiation degree (stratification) for the cells, as keratinocytes, which are non favorable factors for these devices.
In the literature, there are many studies carried out in animals, which show that the presence of cells in an implant is beneficial for the healing of a cutaneous injury and that this benefit is higher if the cells are autologous. Most of the commercial products use heterologous fibroblasts from neonatal foreskin. The selection of this type of cells is due to the easiness for growing them in vitro. However, these cells are heterologous, what additionally implies a rejection risk, the needing of carrying out expensive analysis for guarantying that they are not contaminated with microorganisms, such as HIV or with C hepatitis virus, among others. Additionally, the products comprising only fibroblasts, may influence on a non desirable healing, from an aesthetic point of view as for the fibroblasts may be differentiated into myofibroblasts, responsible of the non desirable contraction of the wound.
Besides products are known wherein autologous dermal and epidermal cells are used. Nevertheless, these are products that distribute the cells in different layers, on a same support, in different faces of the support or in different supports. Generally, said products, are difficult of handling and the availability timing for applying the device is of more than 24 hours, since it is necessary to seed, attach and culture the cells over the surface of the supports or over the cultured dermal cells.
A clear disadvantage of the devices for grafting is the need of adhesives for fixing the implants onto the injuries. Mechanical devices or organic polymers can be used as attachers (stapples or gauze), wherein the main objective of these is to achieve the graft take, being this understood as the fixing of the device to the damaged skin, achieving the placement in the site to be treated.
Lyophilized pigskin has been used to immediate treatment of patients after the occurrence of a traumatic event. This is a transitory solution, since it only allows extending the patient life, while it is possible to apply definitive solutions. In addition, solutions have been developed, on the base of materials that help for burns healing. However, they are cell-free solutions, which unlikely can be compared with the complexity and with the contribution and efficiency in the wound healing, which implies an integrated system with cellular components and a biocompatible matrix developed as part of the present invention.
Therefore, the present invention provides an integrated implant system, being this understood as an intimate link among cells-gel-support, wherein can be combined more than one type of dermal or epidermal, autologous, allogenic, xenogenic or chimeric cells, exhibiting favourable: handling characteristics, time for obtaining the same, application availability, adherence, almost no rejection by the patient, better cellular development and easy conservation. Further, it is informed an easy and fast process for obtaining these integrated implants, wherein the cells are incorporated in the support matrix, providing a device for immediately being applied or conserved.